Pooled adapter strategy for reducing bias in small RNA characterization

ABSTRACT

Modified nucleic acid adapters are provided that collectively provide a mixture of nucleotides at the 3′ end of 5′ adapters and at the 5′ end of 3′ adapters such that at least one adapter in each set has any given nucleotide at position 1, i.e., the nucleotide position available for ligation to a small RNA, and has any given nucleotide at position 2 adjacent to position 1 for use in overcoming bias during nucleic acid manipulation, such as small RNA characterization and/or profiling by, e.g., deep sequencing, along with methods for use of the modified adapters in small RNA characterization. The modified adapters have at least two mixed nucleotides at the adapter terminus to be ligated to a nucleic acid such as a small RNA.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.61/486,214 filed on May 13, 2011.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The disclosure relates generally to the field of nucleic acid sequencedetermination and, more specifically, to deep sequencing approaches toparallel sequence determinations of nucleic acids, e.g., small RNAs,microRNAs, piRNAs.

2. Description of the Relevant Art

Small RNA sequencing (sRNA-seq) is now the gold standard for small RNAprofiling and discovery in fields such as the study of biomarkers incancer, differentiation in stem cells and transposon silencing in thegermline. Biases in deep sequencing which affect the profiling have beenreported, but their etiology has not been identified.

The advent of deep sequencing has now made it possible to sequence thefull complement of small RNAs in a cell. Small RNAs (15-30 nucleotidesor nts) play an important role in a variety of cellular processes.MicroRNAs, piRNAs and endogenous siRNAs are among the many small RNAsthat are crucial regulators of genetic activity. Small RNA profilingthrough deep sequencing has become important for understanding thebiology of these genes and for identifying miRNA markers for diagnosticand therapeutic uses. Real-time PCR (RT-PCR) and microarrays can be usedto profile known small RNAs but a better approach to identifyingdifferences between closely related microRNAs, and to the discovery ofnovel sequences, is deep sequencing. Subtle variations, such as found inisomers, and modifications, such as uridylation, cannot be detected bymicroarrays or RT-PCR. In addition, microarrays and RT-PCR also sufferfrom cross-hybridization artifacts. Deep sequencing is especiallyattractive for its sensitivity to low abundance transcripts. In light ofthese benefits of deep sequencing, a persistent mystery in the field ofsmall RNA sequencing is the discrepancy between the results from deepsequencing, microarrays and qPCR.

Accordingly, a need continues to exist in the art for unbiased methodsof efficiently and accurately profiling the RNAs of a biological samplesuch as a cell. A need also continues to exist for deep sequencingtechniques that show a reduced, or absent, bias in sequenced RNAs.

SUMMARY OF THE INVENTION

Accurate quantification of nucleic acids using deep sequencing isundermined by any bias introduced during the processing or manipulationof those nucleic acids during characterization and, in the case of smallRNA characterization such as microRNA (miRNA) or piwi RNA (piRNA)characterization, the particular sRNA (e.g., miRNAs) could be under orover-represented. This would call into question quantitative data fromdeep sequencing, especially when used to assess the relative abundanceof isoforms and variants. Although biases might also affect otherprofiling platforms, the potential presence of these biases in small RNAsequencing, or sRNA-seq, would undermine the incredible sensitivity andaccuracy made possible by deep sequencing. For piRNAs, sequence featuressuch as the Tbias at the 5′ end are obtained by profiling the nucleotidebias at each position of the sequence. This information can provideclues to the biogenesis of these piRNAs. A data set that is biased bycollection methods can, therefore, lead to erroneous conclusions.Accordingly, the disclosure identifies the problem of bias in nucleicacid manipulation, e.g., nucleic acid derivatization with adapters andnucleic acid amplification, such as would be found in characterizingsRNA.

The disclosed subject matter provides materials and methods forcharacterizing nucleic acids, including characterizing nucleic acidpopulations. For example, the disclosure provides materials and methodsfor preparing nucleic acids to be deep-sequenced, such as by preparingcellular RNAs for deep sequencing. The materials and methods of thedisclosure exhibit reduced or non-existent bias in terms of RNAsamenable to manipulation, including the ligation of terminal adapters toRNAs and the amplification of such RNAs by any means known in the art.The sequencing of small RNAs, i.e., sRNA-seq, typically requires aseries of isolation, ligation and amplification steps to prepare thesRNA of a cell or tissue into a library for sRNA-seq, or sequencing.Each of these steps involves some loss of material and, consequently,the sensitivity and accuracy of the sequencing decreases. Uponinvestigation of each of these steps, it has been discovered that areproducible discrepancy can arise in the ligation or amplificationsteps.

The most widely used technique of sRNA-seq involves the addition of 3′and 5′ adapters onto the ends of the small RNAs by direct RNA ligationusing T4-RNA ligases (Rnl2 and Rnl1 respectively, FIG. 1), followed byan amplification step. Each of the steps in sample preparation resultsin some loss of material, but it has been assumed that these losses arenon-specific. If there were any biases introduced in the ligation steps,however, it would result in errors that are not easy to correct,especially since RNA ligases are known to have biases that have not beenexplored in the context of their use in deep sequencing. Thus, disclosedherein is a systematic investigation of the presence and source ofbiases in sRNA-seq. Small RNAs from 293T human kidney-derived cells andmouse embryonic stem (mES) cells were deep sequenced, using strategiesaimed at identifying the source(s) of bias(es). The data reveals that areproducible discrepancy can arise in the ligation or amplificationsteps. Specifically, the T4-RNA ligases used in sample preparation arethe predominant causes of distortions arising during RNA ligase-mediatedsequence-specific ligations. These biases can be overcome using a pooledadapter strategy. The data provides a basis for new insight into theefficiency of RNA-ligases through deep sequencing, and provides aninvaluable strategy to reduce biases in RNA libraries and thereby obtaina more accurate profile of the small RNA transcriptome.

To address the issue of bias, sequence biases in ligase activity wereexamined and characterized, revealing the consequences of these biaseson the results of sequencing. Through a comprehensive series of smallRNA sequencing experiments, using sequencing adapters with differenttermini and miRNA samples from different tissues, it has beenestablished that the RNA-ligases have strong sequence-specific biases,which significantly distort the miRNA profiles. In response to thesefindings, a pooled adapter strategy has been devised that overcomes thisbias. A comparison of the sequencing data to microarray and qPCR datafurther establishes the accuracy of this method. In light of the resultsdisclosed herein, published small RNA profiles as well as barcodingstrategies using adapter-end modifications, are expected to bemisleading due to the effect of bias. By providing a wide spectrum ofsubstrate for the ligase, the pooled-adapter strategy disclosed hereinprovides a means to overcome issues of bias, and to generate moreaccurate small RNA profiles.

Various aspects of the disclosed subject matter are described in thefollowing paragraphs.

A set of pooled 5′ RNA adapters for small RNA characterization iscomposed of a plurality of adapter bases that are covalently extended atthe 3′ ends by addition of a nucleotide (nt 1), wherein covalently boundnt1 is selected from the group consisting of guanylate, adenylate,uridylate, cytidylate, inosine monophosphate, and 5-bromouridylate,thereby yielding a set of pooled 5′ RNA adapters. In some embodiments,the 3′ nucleotide is selected from the group consisting of guanylate,adenylate, uridylate and cytidylate. Each adapter of unique sequence maybe present in about equimolar concentration relative to other adaptersin the pool.

In some embodiments, the pooled 5′ RNA adapters also include anucleotide 2 (nt2) covalently bound 3′ to nt1, wherein the covalentlybound nt2 is a nucleotide selected from the group consisting ofguanylate, adenylate, uridylate, cytidylate, inosine monophosphate, and5-bromouridylate. Additionally, a nucleotide 3 (nt3) may be covalentlybound 3′ to nt2 and a nucleotide 4 (nt4) covalently bound 3′ to nt3,wherein each of covalently bound nt3 and nt4 is a nucleotide selectedfrom the group consisting of guanylate, adenylate, uridylate,cytidylate, inosine monophosphate and 5-bromouridylate. In someembodiments, each adapter comprises a PCR primer binding site. In otherembodiments, each adapter comprises a restriction endonuclease cleavagesite.

In an embodiment, a set of pooled 3′ adapters for small RNAcharacterization is composed of a plurality of adapter bases that arecovalently extended at the 5′ ends by addition of a nucleotide (nt 1),and wherein the covalently bound nt1 is selected from the groupconsisting of deoxyguanylate, deoxyadenylate, thymidylate,deoxycytidylate, guanylate, adenylate, uridylate, cytidylate,deoxyinosine monophosphate, inosine monophosphate,deoxy-5-bromouridylate and 5-bromouridylate. The pooled 3′ adapters, insome embodiments, may be pooled 3′ DNA adapters. Each adapter of uniquesequence may be present in about equimolar concentration relative toother adapters in the pool.

The pooled 3′ DNA may also include a nucleotide 2 (nt2) covalently bound5′ to nt1 and wherein covalently bound nt2 is a nucleotide selected fromthe group consisting of deoxyguanylate, deoxyadenylate, thymidylate,deoxycytidylate, deoxyinosine monophosphate, and deoxy-5-bromouridylate.In some embodiments, a nucleotide 3 (nt3) may be covalently bound 5′ tont2 and a nucleotide 4 (nt4) may be covalently bound 5′ to nt3, whereineach of covalently bound nt3 and nt4 is a nucleotide selected from thegroup consisting of deoxyguanylate, deoxyadenylate, thymidylate,deoxycytidylate, deoxyinosine monophosphate and deoxy-5-bromouridylate.In some embodiments, each adapter comprises a PCR primer binding site.In other embodiments, each adapter comprises a restriction endonucleasecleavage site.

In an embodiment, a kit for amplifying small RNA includes a set ofpooled 5′ DNA adapters and a set of pooled 3′ adapters, as describedabove. In one embodiment, the set of pooled 3′ adapters is a set ofpooled 3′ DNA adapters. The kit may also include a T4 RNA Ligase 1(Rnl1) and a T4 RNA Ligase 2 (Rnl2). The kit may also include atruncated T4 RNA ligase 2.

A method of profiling small RNA in a sample includes:

-   -   (a) contacting the sample with a set of pooled 5′ DNA adapters        and a set of pooled 3′ adapters as described above;    -   (b) ligating the adapters to the small RNA in the sample;    -   (c) amplifying the ligated small RNAs; and    -   (d) detecting amplified small RNAs, thereby profiling the small        RNAs.

In some embodiments, the small RNAs in the sample are contactedseparately by the pooled 5′ DNA adapters and pooled 3′ DNA adapters. Thesmall RNAs may be contacted by the pooled 3′ DNA adapters in the absenceof ATP.

The 5′ RNA adapters may ligated to the small RNAs using Rnl1 RNA Ligase.The 3′ adapters may be ligated to the small RNAs using Rnl2 RNA Ligase.Ligating the 3′ adapter to small RNAs may be mediated by truncated T4RNA Ligase 2.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention will become apparent to thoseskilled in the art with the benefit of the following detaileddescription of embodiments and upon reference to the accompanyingdrawings in which:

FIG. 1 depicts a protocol for preparing samples for small RNAsequencing;

FIG. 2A depicts a histogram of mi RNA abundance for miR-18a microRNAs;

FIG. 2B depicts a histogram of mi RNA abundance for miR-20a microRNAs;

FIG. 2C depicts a histogram of mi RNA abundance for miR-106b microRNAs;

FIG. 2D depicts an analysis of the effect of PCR cycles on the results;

FIGS. 3A and 3C depict the fraction of miRNA measured by each adaptertype, in 293T and mES samples, respectively;

FIGS. 3B and 3D depict the differences that arise from variations in theefficiencies that depend on the miRNA-adapter combination in 293T andmES, respectively;

FIGS. 4A and 4B depict the effect of the two terminal 3′ nts of the 5′adapter on T4-RNA ligase 1 (Rnl1) ligation efficiency;

FIG. 5 shows that synthetic RNA ligation to a 3′ adapter is enhanced byusing a pool of 3′ adapters with random NN at the 5′ end;

FIG. 6 depicts fluctuation plots showing ligation efficiencies fordifferent fNN (A, C panels) and eNN (B, D panels) adapters against themost abundant miRNAs from 293T (A, B panels) and mES (C, D panels)cells;

FIG. 7 depicts a comparison of parameters inferred from fNN (left panel)and eNN (right panel) against fNN eNN data;

FIG. 8 depicts a radar plot showing the performance of different adaptertermini combinations (fNN eNN);

FIG. 9 depicts a comparison of sequencing against microarray (panels Aand B) and RT-PCR (panels C and D) for mES (panels A and C) and 293T(panels B and D); and

FIG. 10 shows a comparison of rankings between the standard adapters(noNN, ranks along x-axis) versus fNN eNN (ranks along y-axis) for 293T(left panel) and mES samples.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but to the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood the present invention is not limited toparticular devices or methods, which may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include singular and pluralreferents unless the content clearly dictates otherwise. Furthermore,the word “may” is used throughout this application in a permissive sense(i.e., having the potential to, being able to), not in a mandatory sense(i.e., must). The term “include,” and derivations thereof, mean“including, but not limited to.” The term “coupled” means directly orindirectly connected.

The disclosure provides an approach to the characterization of nucleicacid samples, e.g., cell, tissue or organ small RNAs, that addresses theissue of bias in adapter ligation to small RNAs by providing a pool ofadapters modified at the termini ligated or to be ligated to the smallRNAs. The adapter modifications involve the covalent addition of atleast two or more mixed nucleotides to the ligated or to be ligatedterminus of each 5′ adapter base and each 3′ adapter base. An adapterbase may be any adapter known in the art provided that the compound hasnot yet been modified by terminal addition of mixed nucleotides. Thus,the disclosure provides a set of pooled 5′ adapters containing at leasttwo mixed nucleotides at the 3′ termini available for ligation to the 5′end of small RNAs. Also provided is a set of 3′ adapters containing atleast two mixed nucleotides at the 5′ termini available for ligation tothe 3′ end of small RNAs. By mixed nucleotide is meant more than onenucleotide available for incorporation into the set of adapters at agiven position. For the 5′ adapter, at least the two nucleotidepositions at the 3′ end of the adapter are added using mixednucleotides, creating a set of 5′ adapters containing individualadapters having in common the sequence of the adapter base, but havingat the 3′ end of the sequence any one of the mixed nucleotides atpositions 1 and 2, where position 1 is at the 3′ terminus and position 2is adjacent thereto. The mixed nucleotides typically will contain eachof the four conventional nucleotides appropriate to the type of DNAadapter base (DNA or RNA), but the mixed nucleotides may insteadcontain, or contain in addition, any nucleotide analog known in the art,such as inosine monophosphate, deoxyinosine monophosphate,5-bromouridylate or deoxy-5-bromouridylate. For the 3′ adapter, at leastthe two nucleotides at the 5′ end of the adapter are added using a setof the above-described mixed nucleotides appropriate to the adapterbeing modified (DNA or RNA).

In some embodiments, a pool of adapter bases may include between 1 and25 nucleotide bases. As used herein the term “randomized adapters”refers to adapter bases whose ends are extended by 1 to 25 nucleotidebases that vary by nucleotide composition in a random or arbitrarymanner. The ends of the randomized adapters may be composed of equal orweighted DNA, RNA nucleotides.

In some embodiments, the adapter modification, i.e., the nucleotideaddition to the adapter base, involves one, two, three, four or morenucleotides covalently bound to the 5′ end of a 3′ adapter or to the 3′end of a 5′ adapter. Preferred are modifications comprising one, two,three, four or more covalently bound nucleotides. Preferred nucleotidemodifications are collectively fully mixed in that, for each addedposition in the adapter, each of the four conventional nucleotides(e.g., ribo- or deoxyribo-G, A, U/T and C, as appropriate depending onthe nature of the adapter base (DNA or RNA)) are used in approximatelyequal amounts for synthesis using any technique known in the art. Alsocontemplated are additions to the 3′ terminus of a 5′ adapter and the 5′terminus of a 3′ adapter that have equal or differing numbers of addedmixed nucleotides. Beyond the use of nucleotide that are fully mixed ateach position, the disclosure contemplates individual positions thatare, collectively, partially mixed nucleotides or that are singlenucleotides. In particular, as experience with particular adapters andadapter bases is developed, it is expected that an understanding willdevelop regarding the bias of particular RNA Ligases with respect toparticular adapter base sequences such that the preferred terminalnucleotide(s) are known and can be engineered to be present in eachadapter used in a ligation attaching the adapter to a small RNA.

The disclosure also comprehends the use of unconventional nucleotides innucleotide-based modifications by covalent addition to adapter bases.For example, one or more added positions is occupied by (ribo- ordeoxyribo-) IMP, 5-bromouridylate, and/or other unconventionalnucleotides or nucleotide analogs known in the art. Apparent from thedescription herein, the additions to the adapter bases will be useful inmodifying any of a wide variety of adapter bases and the modificationsby covalent additions are not limited to particular adapter bases oradapter bases of particular nucleic acid sequence(s). It will beappreciated that the adapter bases disclosed herein, see, e.g., Table 1,are adapters of any useful or desired sequence, including adapters knownin the art.

To aid in understanding the detailed description of the compositions andmethods according to the disclosure, a few express definitions areprovided to facilitate an unambiguous disclosure of the various aspectsof the disclosure.

An “adapter base” is a nucleic acid adapter with termini that have notbeen modified in accordance with the present disclosure. An “adapter” or“modified adapter” refers to an adapter base modified by addition of atleast one nucleotide to at least one terminus of the adapter base.Typically, adapters or modified adapters will be covalently linked tosRNA, or the corresponding DNA molecule, or the DNA complement of thesRNA for use in manipulating the construct or characterizing theconstruct or sRNA sequence, or its DNA counterpart or complement.

A nucleic acid “modification” is a chemical alteration to a nucleic acidmolecule. Preferably, a nucleic acid modification is the covalentaddition to one of the two termini of an adapter base. For 5′ adapters,i.e., adapters ligated or to be ligated to the 5′ end of a small RNA,the modification(s) occur at the 3′ terminus of the adapter base toyield the adapter. For 3′ adapters, i.e., adapters ligated or to beligated to the 3′ end of a small RNA, the modification(s) occur at the5′ terminus of the adapter base to yield the adapter. A sRNA joined toadapters will have two bound adapters, and these adapters may be thesame or different. The adapters typically provide functionality usefulin sRNA manipulations to characterize the sRNA, such as including a PCRprimer binding site, a restriction site(s) useful for sizing and/orcloning the sRNA or a product (e.g., cDNA) made therefrom.

An “sRNA” is a small RNA as that term is understood in the art andincludes microRNA (miRNA) and piwiRNA (piRNA). Apparent from their usein context herein, these terms may be used interchangeably. Typically,sRNA molecules are involved in at least one aspect of regulating cellphysiology, such as involvement in the regulation of the expression ofone or more genes.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 discloses the materials and methods used in the experimentsdescribed herein. Example 2 describes many of the experiments reportedherein, including the construction of sRNA libraries using variousmodified adapter bases which may be 5′ or 3′ adapters. Example 3addresses the biases revealed in the experiments and models for reducingor eliminating bias.

Example 1

This Example provides a disclosure of the materials and methods used inconducting the experiments disclosed in the remaining working examples.

ES Cell Culture

R1 mouse ES cell line was cultured feeder-free on 0.1% gelatin-coatedplates in DMEM; Hi-Glucose, 15% fetal bovine serum, non-essential aminoacids, L-glutamine, β-mercaptoethanol, penicillin/streptomycin, sodiumpyruvate and leukemia inhibitory factor (LIF). Cells were grown in ahumidified incubator in 5% C02 and 95% air. These cells were kindlydonated by the Lemischka laboratory at Mount Sinai School of Medicine.

Library Construction and Sequencing

Total RNA was isolated from 293T cells and mouse embryonic fibroblastsusing Trizol extraction (Invitrogen). Sequencing libraries enriched formicro RNAs were constructed using a modified version of a small RNAprotocol detailed by Pfeffer (Pfeffer et al. “Cloning of small RNAmolecules.” Current Protocols in Molecular Biology/Edited by FrederickM. Ausubel . . . et al, Chapter 26 (November 2005), Unit 26.4. PMID:18265364). Two RNA markers were synthesized Spike 19(CGUACGGUUUAAACUUCGA; SEQ ID NO:1) and Spike 24(CGUACG-GUUUAAACUUCGAAAUGU; SEQ ID NO:2) (Sigma Aldrich). RNA wasend-labeled using polynucleotide kinase and radioactive ATP (P32). Tenmicrograms of total RNA was size fractionated by denaturingpolyacrylamide gel electrophoresis (PAGE, 12% gel). miRNAs were excisedfrom the gel using radiolabeled markers as guides. Purified miRNA wasligated to a 17 nt 3′ adapter with truncated T4 RNA ligase 2 (Rnl2) inan ATP-free buffer (BioScientific). The ligated fragment of 36-41 nt wasPAGE purified. A second RNA adapter was ligated to the 5′ side of theproduct using T4 RNA ligase 1 (Rnl1) and buffer containing ATP. The72-78 nucleotide ligated fragment was PAGE purified and then reversetranscribed using a specific primer (BanI-RT; ATTGATGGTGCCTACAG; SEQ IDNO:3). cDNA was amplified by 22 cycles of PCR with primers thatincorporate sequences compatible with the Illumina platform (Sol-5-SBS,AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACG, SEQ ID NO:4 and Sol-3-ModBan,CAAGCAGAAGACGGCATACGATTGATGGTGCCTACAG; SEQ ID NO:5) (FIG. 1). Thelibrary was sequenced using the Illumina Genome Analyzer IIx at36-nucleotide read length.

Microarray

miRNA abundance was assessed in 293T and mES RNA samples byoligonucleotide microarray using Affymetrix GeneChip (miRNA 1.0). One μgof total RNA was labeled using the FlashTag Biotin 3DNA kit(Genisphere), as follows: polyadenylation of RNA by polymerase, ligationto a biotinylated 3DNA molecule mediated by an oligonucleotide with 5′polyd(T) and 3′ 3DNA complementary adapter. Labeled RNA was hybridizedto the microarray using standard Affymetrix methods. Fluorescenceintensities were extracted using the R statistical package, usingmethods from the BioConductor module.

Real-Time PCR

Quantitative real-time PCR was carried out using the Applied Biosystems(AB) microRNA specific reagents and a 7900HT thermocycler. Ten ng oftotal RNA were reverse transcribed with a miRNA-specific hairpin primerusing the AB micoRNA Reverse Transcription kit. Specific forward primersand universal reverse primers were random with cDNA and AB Universal PCRMaster Mix (no UNG) as recommended by the manufacturer. The followingmiRNAs were assayed: hsa-mir-18a, -20a, -106b, -92a, -103-2, -10, -16,-17 and hsa-let-7. Ct values were extracted from real-time data usingthe auto threshold setting.

Computational Analysis

Analysis of such datasets is well established, but extracting theinserts from the libraries was complicated by three causes: (1)sequencing errors that miscall a base, (2) sequencing errors that miss abase, and (3) errors in the synthesis of the NN constructs. To mitigateproblems from sequencing errors, we only accepted sequences where the 3′adapter sequences were matched exactly. This eliminates most of theproblematic reads, but does not solve the issue of point 3 above. Forthat, we used the relative abundances of the various inserts in thesmall RNA library (from our analysis of data from several runs), toidentify synthesis errors. Failure to synthesize a particular N, or askew in a particular N, could give rise to a misidentification of theorigin of the sequence (which library it came from), or itsend-modifications. Each sequence was binned into the appropriate NNcategory, as well as the appropriate version of the miRNA sequence (thecanonical mature or some variant, either derived from the originalhairpin sequence or a non-template modification). Most of the processingwas done using custom Perl scripts. Custom R-scripts were used togenerate the graphs and statistical analyses.

Example 2

In order to establish if the ligation of adapters to small RNAs wassequence dependent, libraries were constructed for small RNA sequencing,by customizing the standard protocol (FIG. 1), using modified adapters.Total RNA is size fractionated by denaturing polyacrylamide gelelectrophoresis (PAGE) and miRNAs are excised from the gel usingradiolabeled markers as guides. Purified small RNAs are ligated, using atruncated T4 RNA ligase 2 (Rnl2) in an ATP-free buffer, to a 17 ntmodified 3′ DNA adapter with a dideoxy nt at the 3′ end and an activatedadenylation at the 5′ end. The dideoxy nt (more specifically, the deoxy3′ carbon of the 3′-terminal nt) prevents selfligation of the adapter,while the truncated ligase prevents circularization of the small RNAinserts. The ligated fragment of 36-41 nt is then PAGE-purified toremove the unligated 3′ adapters. A 32 nt RNA adapter is ligated to the5′ side of the product using T4 RNA ligase 1 (Rnl1). The 72-78 ntligated fragment is PAGE-purified again to remove the unligated 5′adapters. The product is reverse-transcribed using a specific primer andthe resulting cDNA is amplified by PCR with primers that incorporatesequences compatible with a deep-sequencing platform.

Strategies Using Modified Adapters

To understand the exact nature of biases, we devised strategies usingvarious 5′ and 3′ adapters with additions to the ligating ends (3′-endof the 5′ adapter and the 5′-end of the 3′ adapter). We devised sixstrategies involving these adapter pools, as listed below and in Table1.

-   -   1. noNN, uses the standard modban 5′ and 3′ adapters.    -   2. 4-mer pool, the standard modban 3′ adapter, pool of twelve 5′        adapters with 4-mer additions.    -   3. fNN, uses the standard 3′ adapter and a pool of 5′ adapters        generated by adding random NN additions to the 3′ end of the 5′        modban adapter.    -   4. eNN, uses the standard 5′ adapter with a pool of 3′ adapters        that are modified at the 5′ end with NN additions (eNN).    -   5. fNN eNN, uses a pool of 5′ and 3′ adapters with the NN        modifications described in 3 and 4.    -   6. fNNNN, uses a pool of 5′ modban adapters with the addition of        random NNNN to the 3′ end and the standard 3′ adapter.

TABLE 1 Strategy 5′ adapter RNA 3′ adapter DNA noNNACACUCUUUCCCUACACGACGCUCUUCCGAUC CTGTAGGCACCATCAAT SEQ ID NO: 6SEQ ID NO: 7 fNN ACACUCUUUCCCUACACGACGCUCUUCCGAUCNN CTGTAGGCACCATCAATSEQ ID NO: 8 SEQ ID NO: 9 fNNNN ACACUCUUUCCCUACACGACGCUCUUCCGAUCNNNNCTGTAGGCACCATCAAT SEQ ID NO: 10 SEQ ID NO: 11 eNNACACUCUUUCCCUACACGACGCUCUUCCGAUC NNCTGTAGGCACCATCAAT SEQ ID NO: 12SEQ ID NO: 13 fNN_eNN ACACUCUUUCCCUACACGACGCUCUUCCGAUCNNNNCTGTAdGCACCATCAAT SEQ ID NO: 14 SEQ ID NO: 15 4-mer poolACACUCUUUCCCUACACGACGCUCUUCCGAUCWXYZ CTGTAGGCACCATCAAT SEQ ID NO: 16SEQ ID NO: 17

Table 1 shows miRNA sequencing libraries that were generated with theadapter combinations shown. The 4-mer pool is a mixture of twelveadapters, represented as WXYZ (CTAG, GAGT, CCAA, AGCA, AACC, AAGG, TGAC,CGTC, GCTT, GTAT, GGAA, TGTG). All 3′ DNA adapters have a 5′ rAPP and3′ddC modifications to prevent self-ligation and circularization.

5′ Adapter Ligation Efficiency is Sequence Dependent

In order to determine if there was sequence dependent ligation of the 5′adapters, we prepared small RNA samples from 293T cells, using a pool oftwelve 5′ adapters, modified by the addition of 4-mers (TGAC, GAGT,GTAT, CGTC, GGAA, AAGG, GCTT, AACC, CCAA, AGCA, CTAG, and TGTG). Theresults showed significant bias with little similarity between data fromdifferent adapters (FIG. 2). Sequencing libraries were constructed fromtotal RNA derived from 293-T cells, using a pooled set of twelve 5′adapters that had different 4 mer 3′ ends, shown on the x-axis. There isgreat diversity in the capture of individual miRNAs by different 5′adapters (FIGS. 2A, 2B and 2C show data for miR-18a, miR-20a andmiR-106b microRNAs, respectively). FIG. 2C shows an extreme case wheremiR-106b is captured well by only one adapter, ending in AGCA, out ofthe 12 adapters constituting the pool. These data are consistentlyreproduced in other experiments shown in FIG. 4. To isolate the effectof PCR cycles, samples were prepared twice, using 25 (y-axis) and 18(x-axis) cycles of PCR (FIG. 2D). Each point represents a miRNA. Thecorrelation between the two sets of PCR conditions is high (coefficientof 0.95) and the best linear fit to the points is a line of slope 1,indicating that the data are reproducible and PCR is not responsible forthe biases.

We also prepared individual 293T cell samples using one adapter persample, selecting five 4-mer ends (TGAC, CGTC, AACC, GTAT and GGAA). Wefound wide variations in the miRNA profiles, especially for highlyexpressed miRNAs such as hsa-mir-20a and hsa-mir-18. In table 2, we seethat replicates sequenced using different adapters have poor correlationto each other. When the adapters are pooled (Table 3), then there isconcordance between the profiles for the different replicates. Thisindicates that a pooled approach would reduce the effect of biases dueto adapter ligation on the 5′ end.

TABLE 2 B30_TGAC B25_CGTC 293T_AACC B31_GTAT B29_GGAA B30_TGAC 1 0.6640.687 0.679 0.679 B25_CGTC 0.664 1 0.654 0.637 0.644 293T_AACC 0.6870.654 1 0.6 0.666 B31_GTAT 0.679 0.637 0.6 1 0.62 B29_GGAA 0.679 0.6440.666 0.62 1

Table 2 depicts correlations between samples prepared using individual5′ adapters that differ only at the 3′ terminus. The spearman-rankcorrelation is based on the rankings of the miRNA sequences byabundance. The relatively low correlations, between biologicalreplicates of 293T cells, in contrast to the numbers seen in Table 3,suggests that the efficiency of ligation of the adapters to differentmiRNA sequences is quite variable. The row and column names reflect thesamples and the adapter termini used, so B25 TGAC refers to sample B25which uses the adapter ending in TGAC.

TABLE 3 Samples B26 B30 B25 B29 B26 1 0.91 0.96 0.95 B30 0.91 1 0.910.92 B25 0.96 0.91 1 0.97 B29 0.95 0.92 0.97 1

Table 3 shows correlations between biological replicates of 293T cellsamples, prepared using a pooled-adapter strategy. The correlations(spearman-rank) are based on the ranking of the miRNA sequences based onabundances calculated by averaging them over the five adapters listed inTable 1. This indicates that mixed pools of adapters will help overcomethe biases in ligation efficiency.

Nature of Sequence Dependence in the Efficiency of 5′ Adapter Ligation

To identify the biases inherent in the 5′ adapter ligation, two samplesfrom 293T and mES cells were prepared using the fNN strategy. Theresults showed that the profiles measured from the same sample can varywildly for different adapters (FIG. 3). FIG. 3A (293T) and FIG. 3C (mES)depict the amount of miRNAs (y-axis) captured by each adapter (x-axis),suggesting some adapters are more efficient than others. If it were asimple matter of differing efficiencies for different adapters, then themiRNA profiles derived from each barcode should be scaled versions ofeach other. In fact, as shown in FIG. 2B (293T) and FIG. 2D (mES), theprofiles for different adapters are very dissimilar. In FIG. 2B and FIG.2D, the x-axis shows different miRNAs, ranked by their overalloccurrence, which is the sum over all adapters. The y-axis shows, of themiRNAs captured by a particular adapter, the fraction that each miRNAoccupies. It is apparent there can be dramatic shifts in the rankingsfor the miRNAs (the profiles) between adapters. We wanted to establishhow much of the sequence proximal to the ligating end of the adapterdetermined the ligation efficiency. For this, we carried out anexperiment using 5′ adapters with 4 terminal random nucleotides, thefNNNN strategy. FIG. 4 shows that most of the ligation efficiency can beexplained by the last two nucleotides. Only in one case, hsa-miR-106b,were the four nucleotides needed to pick out an abundant miRNA (see alsoFIG. 2 and FIG. 10).

Biases in 3′ Adapter Ligation

We decided to investigate the bias on the 3′ side, especially since the3′ adapter ligation is very different from the 5′ adapter ligation. Asexplained in Example 1, it involves a truncated RNA-ligase (rn12) thatneeds an adenylated 3′ adapter to carry out the ligation and the adapteris DNA.

We designed a simple gel-shift-based assay to test for the existence ofa 3′ adapter ligation bias. We chose two radioactively labeledoligomers, a 19-mer (CGUACGGUUUAAACUUCGA-SEQ ID NO: 18) and, a 24-merthat had a 5-mer (AAUGU) addition at the 3′ end of the 19 mer. The RNAmarkers were 5′ end-labeled with ³²P and then ligated in duplicate toone of two sets of adenylated 3′ DNA adapters. One set of 3′ DNAadapters consisted of the standard (terminally unmodified) adapter witha 5′ CTGT and the second set consisted of a mixture of adapters thatdiffer from the standard adapter in having two extra mixed base (i.e.,mixed nt) positions on the 5′ side, with the start now becoming 5′NNCTGT. After ligation, the RNA-DNA products were size-fractionated on a12% polyacrylamide gel.

FIG. 5 shows that with the standard 3′ adapter, the 24-mer does not showgood ligation, while the 19 mer shows good ligation, on the other hand,we have good ligation to both oligos with the eNN strategy.Specifically, the 19 nt marker ligated efficiently, irrespective of theadapters used (lanes 5-8), while the ligated 24-mer product is low inabundance when the standard adapter is used (lanes 1-2), but isefficiently ligated (with abundant products) when the mixed-basesadapters are used (lanes 3-4). This suggests two things, the 24 mer hasgood ligation only to certain members in the eNN pool, and that using apool of adapters is better. While this may appear to be crude, thedramatic effect seen in the gel-shift suggests large differences inligation efficiencies between different pairs of sequences, indicatingthat both the 5′ and 3′ adapter biases need to be taken into account inany sequencing experiment using T4-RNA ligases in the samplepreparation.

We pursued a strategy similar to the case of 5′ adapter ligation, usingthe eNN strategy for 3′ adapters in order to systematically study thebiases in the ligation of the 3′ adapter. In FIG. 6, we show theefficiencies of the 5′ and 3′ adapter ligations in the form of afluctuation graph. Fluctuation plots showing ligation efficiencies fordifferent fNN (FIG. 6A, 6C) and eNN (FIG. 6B, 6D) adapters against themost abundant miRNAs from 293T (FIG. 6A, 6B) and mES (FIG. 6C, 6D)cells. “fNN” is defined in the description of FIG. 3. “eNN” refers tothe addition of a mixed nt (A, G, U, C) to the two nucleotide positionsat the 5′ end of a 3′ adapter, i.e., the end of the 3′ adapter ligatedto an sRNA. The naming convention of miRNAs in all the figures definesthe beginning and end of the sequence followed by an “m” (for acanonical mature) or “n” for a non-canonical miRNA sequence followed bythe name of the miRNA. The area of the dark rectangles depicts the valuefor each combination of miRNA and adapter. The standard adapter ends (TCat the 3′ end in fNN and CT at the 5′ end in eNN, highlighted in grayboxes) are not efficiently ligated to the most abundant miRNAs. Even themost efficient adapters show variability, indicating that no singleadapter can be expected to work well across all possible sequences. Forthe most abundant miRNAs, most of the variability comes from the 3′adapter ligation (the eNN adapters, panels B, D). In mES cells, thereare two isomers of mmu-miR-292-3p, the GT-ending 3′ adapter captures theGAGT-ending isomer more efficiently, while the GA-ending 3′ adaptercaptures the GAGTG-ending isomer more efficiently.

The 3′ adapters show more variability, which is probably due to thegreater diversity in the 3′ ends of the miRNAs compared to the 5′ ends,suggesting that the 3′ adapter ligation might be a bigger source ofbiases in measurements. It is interesting to note that the efficiency ofthe standard modban adapters (the 5′ one ends in TC and the 3′ onestarts with CT) is low, compared to some of the others, but there is nosingle adapter that is uniformly efficient across the miRNAs that wetested here. This again suggests that it is necessary to take a pooledapproach on both adapters for an unbiased measurement.

A Model for Ligation Efficiencies

In order to develop a unified picture of the ligation efficiency andshow that the experiments are consistent with each other, we developed amodel. We propose a model for ligation efficiency based on the 256combinations at each ligation junction, determined by the twonucleotides (16 possible combinations, AA, AC . . . TG, TT) on theligating end of the adapter and the two nucleotides (16 possiblecombinations) on the ligating ends of the miRNA. We define these as Fij(i and j each varying from 1 through 16 where 1 stands for AA, 2 for ACgoing on to 15 for TG and 16 for TT) for the 5′ adapter ligation, andEmn (m and n each varying from 1 through 16) for the 3′ adapterligation. Let Mk be the actual abundance of a miRNA labeled k in thesample. Let mk be the measured amount of miRNA labeled k using adapterswith ends i and n on the 5′ and 3′ adapter respectively. Then, thefollowing equation must hold true: m^(k)=F_(ij)*M^(k)*E_(mn).

The various adapter combinations are in equimolar concentrations; sothey do not enter the equation (other than a constant that can beabsorbed in F and/or E). FIG. 7 depicts a comparison of parametersinferred from fNN (left panel) and eNN (right panel) against fNN eNNdata. The rows are miRNAs captured by different methods, alternate rowsare data from the INN eNN. In the figure fTC eNN means the f end was thestandard (TC) and the e end varied, while INN eCT means e end had a CTand the f end varied. In the data for INN eCT versus fNN, the ratio tothe AG-CT combination is depicted for each row. For the comparison offNN eNN against eNN, the ratio of the values for the TC-GT combinationwas considered. The pairs are highlighted (either light- or dark-shadedrectangles), and the numbers between members of a pair are expected tobe similar. There is a striking similarity between pairs of rows,indicating that the fNN eNN parameters are in concordance with separatemeasurements of parameters with fNN and eNN. An explanation for themodel on which the calculations were based is provided herein below.

If this model is universal, we expect that the ratio between various F's(and various E's) from the fNN and eNN datasets should agree with thenumbers derived from fNN-eNN. Since we do not know the Mk for a miRNAlabeled k, we have to eliminate that from any quantity we measure. To dothis, we pick the same value for eNN (CT) in the fNN eNN set as the 5′end on the 3′ adapter in the fNN set. Within each experiment, we thendefine:r _(ia) ^(k) =m ^(k) _(u) /m ^(k) _(aj) =F _(ij) /F _(aj)  (2)r_(ia) ^(k), which is the ratio between the number of miRNA k, capturedby adapters with ends i and a, is now independent of Mk and it should beidentical for the fNN eNN (with eNN set at CT) and the fNN sets. We cando a similar comparison between the fNN eNN and the eNN sets. Theseratios, derived from independent experiments, are shown in thefluctuation plot in FIG. 7. The numbers agree across miRNAs and acrosssample types, suggesting a level of universality for this model. Thesuccess of the model points to the reproducibility of the effects wehave observed. Note, however, that miR-106b shows a bias that depends onthe 4 nt at the 3′ end of the 5′ adapter (panel FIG. 2D and FIG. 4D).Strategy to Overcome the Ligation Biases

Based on all the evidence presented above, we devised the fNN eNNstrategy, described at the beginning of this section, to overcome thebiases. We generated libraries for sequencing small RNA from the 293Tand mES cells. FIG. 8 depicts a radar plot showing the performance ofdifferent adapter termini combinations (fNN eNN), shown outside thecircle in blue. The inner circles represent percent contribution of eachadapter combination to a particular miRNA that was sequenced. Data arepresented for the top miRNA (hsa-miR-20a) in 293T cells and for the twotop miRNAs (mmu-miR-292-3p and mmu-miR-294) from mouse embryonic stemcells. There was significant variation in the efficiency of capturebetween various combinations of 5′ and 3′ adapter end modifications. Thedata are consistent with adapter pooling, e.g., using the fNN eNNstrategy, reducing the amount of starting material needed for sequencingsmall RNAs. Each microRNA seems to have a favored fNN-eNN pair thatworks best, once again suggesting the need for a pooled adapterapproach.

Validity of the fNN eNN Strategy

Our proposed strategy, fNN eNN, is one that optimally picks up mostsequences, and can help overcome the effect of the biases and increasethe efficiency of small RNA sequencing. FIG. 9 depicts a comparison ofsequencing against microarray (FIGS. 9A and 9B) and RT-PCR (FIGS. 9C and9D) for mES (FIGS. 9B and 9D) and 293T (FIGS. 9A and 9C). There wereoutliers, such as miR-106b, that were only captured by the fNNNNstrategy, but overall, there was significant correlation between the fNNeNN strategy and the microarray data (FIG. 9A) and the fNN eNN strategyand the RT-PCR data (FIG. 9C), while the fNN sequencing strategy doesnot correlate as well to RT-PCR and array data (FIG. 9B and FIG. 9D).

Using the fNN eNN technique, we have identified several miRNAs in mouseembryonic stem cells and human kidney derived 293T cells that areseverely underrepresented in the current published profiles based ondeep-sequencing. FIG. 10 depicts a comparison of rankings between thestandard adapters (noNN, ranks along x-axis) versus fNN eNN (ranks alongy-axis) for 293T (left panel) and mES samples (right panel). A pointabove the diagonal represents a sequence that was over-represented innoNN, while below the diagonal are points that were under-represented innoNN. The hsamiR-18a was over-represented in the noNN case, where it wasranked 3, the array and qPCR data agreed better with the fNN eNN resultswhich ranked it much lower (this skew was also seen in the mES samples,but the ranking in the noNN was 22 while the fNN eNN was much lower(135). In the mES sample, mmu-miR-294 was first and a non-canonical formof mmumir-292-3p was second for noNN. While they switched ranks in thefNN eNN case, the difference is very significant because the abundancesof the first and the second ranks were about 2-fold apart, indicating astrong bias. mmu-miR-290-5p was very high at rank 5 in the case of noNN,it was outside the range of the graph in fNN eNN, in accordance with theqPCR data. Thus, in every case in which a difference was detectedbetween noNN and fNN eNN, fNN eNN was more accurate in reflecting theprofiles.

Thus, we have established the existence of a pronounced,sequence-dependent bias in the ligation of 5′ and 3′ sequencing adaptersto miRNAs. Our proposed strategy, fNN eNN will be able to overcome thelimitations of the bias in the RNA-ligase and make sRNA-seq morerepresentative of the profiles in the underlying samples.

Example 3

Our experiments provide an understanding for the biases observed withsRNA-seq. We have identified and quantified biases in the functioning ofthe T4-RNA ligases (Rnl1, Rnl2) through deep sequencing, and the largenumbers of ligated sequences generated here provide a measure ofstatistical reliability to our results. “Deep sequencing” is used hereinin conformity with the ordinary meaning of the term in the art, i.e.,high-throughput sequencing methodology such as the massively parallelsequencing methodologies of lumina and 454.

Reasons for Biases in the Ligase Activity

Bacteria, under viral attack, nick their tRNAs to block translation. TheT4-phage uses the ligases to repair the nick. Since the nicks are madeat specific sequences in the tRNAs, the ligase structure most likelyhave evolved sequence-specificity to efficiently repair the nicks.

Profiling Studies

Our studies have important implications for profiling studies ongoing inthe fields of genomic profiling of small RNAs, such as studies in cancerand stem cells, that attempt to identify bio-markers for diagnosis andtherapies.

Even a small change in the ranking, from say 1 to 2, reflects a bigchange in numbers. Thus a small change in the profile is a profoundchange in the underlying numbers, which can have important implicationsfor the kinetics of the reactions mediated by the miRNA.

There are several mES-specific miRNAs. The microRNA, mmu-miR-292-3p, hastwo forms, a canonical form and a longer non-canonical form with anextra A at the 5′ end. This suggests that the two forms have differenttargets. Thus, it is important to understand their relative abundancesas it might have important implications for stem-cell biology. In thenormal protocol, with the standard adapters, the canonical form isranked second, while the non-canonical form is about one-third asabundant (1,305,991 versus 552,573). In the fNN eNN strategy, the twoare the highest ranked, with the canonical form ranked first and thenon-canonical form ranked second and about two-thirds as abundant(3,085,673 versus 2,356,385). The microarray ranks both as the mostabundant miRNAs, but it probably cannot distinguish between the twoisoforms.

The Model

The ability of a unified model to predict the outcomes of samplepreparations using different adapters suggests that the effects are notstochastic. From the model that we have discussed for the ligation bias,it might appear that a single set of 5′ and 3′ adapters might suffice,allowing for mathematical corrections to be applied to the profiles thatare derived. This is illusory, since, for every adapter, we see at leastone transcript that seems to be inefficiently ligated. Applying largecorrections can result in excessive noise, reducing the reliability ofthe corrected results. Thus, in applications where it is critical toestablish accurate profiles, using pooled adapters of fNN eNN strategymight be the best approach. We have made a persuasive case for thisthrough our series of experiments.

Explaining the Gel-Shift Experiment

We can derive an equation similar to Equation 2, for the ratios ofactual measures values for the members of these pairs. In order toexplain the gel-shift experiment, we have to pick eNN data for twomiRNAs that have the same 3′ terminus as the 19-mer (GA) and the 24-mer(GT), and identical 5′ termini. The pair, (TATT-CTGT.m.h-92a,TAGC-TTGA.m.h-21) satisfy the requirements for the ends of thesequences. The ratio of the measured values divided by the ratio of theestimated values of the miRNAs in the sample (using estimates from thefNN eNN case), is the ratio of ligation efficiencies. For the pair,(TATTCTGT. m.h-92a, TAGC-TTGA.m.h-21) the ratios of the ligationefficiencies works out to 0.53, so we would predict that the 19 merwould be twice as efficiently captured as the 24 mer. It is tough toestimate these numbers accurately, as the abundances of the two miRNAsin the pair is not very high.

fNNNN Strategy

The miRNAs, mir-106b and mir-20a are identical at the first 9nucleotides on the 5′ end. Despite this, mir-106b is efficientlycaptured only in the fNNNN strategy by a few adapters (as we alreadydiscussed in FIGS. 2,4), but the fNN strategy does not capture mir-106bvery efficiently. In contract, mir-20a is efficiently captured by boththe fNNNN and the fNN strategies. This indicates that there might beother factors such as secondary structures that could influence theligation. It is also of biological interest to identify the distinctroles of the two miRNAs (especially as mir-20a seems to be abundant inmany tissues) and if the inefficiencies in capturing mir-106b has led toits role being overlooked.

miRNA Clusters

It is believed that all members of a miRNA cluster (miRNAs that are inclose proximity (<1 Kb apart from each other) are processed from asingle transcriptional unit, in which case, differential expressionpatterns within a cluster implies differential regulation. Thus,accurate measurement of the relative numbers for members of a cluster isbiologically very relevant. We can extract numbers for two clusters,(miR-106b, miR-93, miR-25) that we label the 106b cluster and (miR-17,miR-18a, miR-19a, mir-92a, mir-19b, miR-20a) that we label the mir17cluster. Depending on the strategy used, the relative amounts withineach cluster are different. For the 106b cluster, the numbers(fold-change) relative to the miR-106b abundance are, noNN (1.0, 4.8,1.6) and fNN eNN (1.0, 9.4, 1.95), there is a big change in the relativeabundance of miR-93. For the mir17 cluster, the numbers (fold-change)relative to the miR-17 abundance are, for noNN (1.0, 1.19, 0.32, 0.70,0.50, 3.39) and for fNN eNN (1.0, 0.45, 0.26, 0.80, 0.50, 2.30), thereis a big change in the relative abundance of miR-18a. Thus, depending onthe technique used, we have a different interpretation for the miR-17abundance.

Abundance of Star Sequences

In microRNAs, the star sequence is usually degraded and not found in thesequencing data, but occasionally, for certain abundant miRNAs, the starsequence is also captured. A question that often arises is how abundantthe star sequence is and if it has any function. In the case of miR-17in 293T cells, we find two star forms, the canonical one (*) and a formwith an extra C at the 3′ end (*C). The relative numbers of the starforms (*,*C) versus the mature for different techniques are, fNN eNN(0.176, 0.2) and noNN (0.0672, 0.23). The star sequence abundance isstrongly dependent on the sequencing method.

piRNA Sequencing

piRNAs are small RNAs, 28-32 nt long, that are exclusively expressed inanimal gonads. They are involved in transposon control and germlinemaintenance. Deep sequencing has been used to analyze piRNAs, mainlyfrom Drosophila and mouse. A distinguishing feature of a particularclass of piRNAs (primary piRNAs) is the bias for a T at position 1, anda change in this bias is considered an indication for piRNA processingdefects. In a particular experiment, small RNA libraries were generatedfrom wildtype and mutant samples using 5′ adapters with same ends (TC).The resultant sequence sets showed >80% T-bias. However, generation ofbiological replicates from additional mutant samples, but now using 5′adapters with different 3′ ends, resulted in alarmingly varyingT-biases: 73% (for an adapter ending in GA), 69% (TA), and 57% (AA).This indicates that a careless choice of adapters can give rise toerroneous conclusions, even comparisons between libraries generated withthe same adapters might come with problems, due to the changes in thesmall RNAs which might result in different efficiencies of adapterligation.

Practical Implications

The practical implications of these studies are that:

-   -   Profiling by sequencing needs to be done using pools of adapter        sequences.    -   Isomer profiles generated using a single adapter sequence need        to be revisited.    -   Many studies have reported end-modifications of mature        sequences, such as, uridylation. The modifications might have        been under- (or over-) reported, because of the biases in the        activity of the ligases.    -   The isoforms identified as mature in mirBase are usually the        dominant ones, which may reflect the biases of the profiling        methods rather than their natural biological enrichment.    -   Barcoding of samples using adapters, for multiplex sequencing,        should be done carefully, taking these results into account. It        is expected that by using the methods according to the        disclosure, bias in barcoding can be reduced or eliminated.

The studies disclosed herein have established that RNA ligases derivedfrom T4-phage exhibit significant sequence-specificity in theiractivity. The profiles of small RNAs are strongly dependent on theadapters used for sample preparation. In light of this, the current,popular, sRNA-seq protocols need revision. The disclosure provides thatrevision in disclosing materials and methods useful in covalentlyligating at least 2 nucleotides to the 3′ end of a 5′ RNA adapter and/orat least 2 nucleotides to the 5′ end of a 3′ nucleic acid adapter thatis either RNA or DNA, with the ligations mediated by an RNA ligase suchas T4 RNA Ligase 1, T4 RNA Ligase 2, or truncated T4 RNA Ligase 2. Wefind that a mix of adapters, with different sequence ends, permits amore accurate estimation of the amounts of individual nucleic acids,such as miRNA sequences and their isoforms.

In this patent, certain U.S. patents, U.S. patent applications, andother materials (e.g., articles) have been incorporated by reference.The text of such U.S. patents, U.S. patent applications, and othermaterials is, however, only incorporated by reference to the extent thatno conflict exists between such text and the other statements anddrawings set forth herein. In the event of such conflict, then any suchconflicting text in such incorporated by reference U.S. patents, U.S.patent applications, and other materials is specifically notincorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the invention may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description of the invention.Changes may be made in the elements described herein without departingfrom the spirit and scope of the invention as described in the followingclaims.

What is claimed is:
 1. A mixture of 3′ DNA adapters for small RNAcharacterization comprising: a plurality of modified adapter sequencesthat are covalently extended at the 5′ ends by addition of at least tworandom nucleotides, wherein the modified adapter sequences comprise thestructure NNe, wherein e is the adaptor sequence, and wherein NN are atleast two random nucleotides that have been covalently attached to the5′ end of the adapter sequence; wherein at least one modified adaptersequence further comprises an activated riboadenylation (rAPP) at the 5′end of the modified adapter sequence, wherein the at least two randomnucleotides are internal to the activated riboadenylation(5′rAPP-NNe-3′); wherein the mixture comprises at least sixteen modifiedadapter sequences for each unique adapter sequence wherein the at leastsixteen modified adapter sequences comprise AAe, ACe, AGe, ATe, CAe,CCe, CGe, CTe, GAe, GCe, GGe, GTe, TAe, TCe, TGe, and TTe; and wherein Ais a deoxyadenylate nucleotide; C is a deoxycytidylate nucleotide; G isa deoxyguanylate nucleotide; and T is a thymidylate nucleotide.
 2. Themixture of 3′ DNA adapters according to claim 1, wherein the 3′ adaptersare 3′ RNA adapters.
 3. The mixture of 3′ DNA adapters according toclaim 1, wherein at least one modified adapter sequence furthercomprises a restriction endonuclease cleavage site.
 4. The mixture of 3′DNA adapters according to claim 1, wherein each adapter sequence ofunique sequence is present in about equimolar concentration relative toother adapter sequences of unique sequence in the mixture.
 5. Themixture of 3′ DNA adapters according to claim 1, wherein at least onemodified adapter sequence further comprises a PCR primer binding site.6. The mixture of 3′ DNA adapters according to claim 1, wherein at leastone modified adapter sequence comprises: deoxyinosine;deoxy-5-bromouridylate; or deoxyinosine and deoxy-5-bromouridylate. 7.The mixture of 3′ DNA adapters according to claim 1, wherein at leastone modified adapter sequence further comprises a dideoxy nucleotide atthe 3′ end of the modified adapter sequence.
 8. The mixture of 3′ DNAadapters according to claim 1, wherein at least one 3′ adapter comprisesribonucleotides.
 9. The mixture of 3′ DNA adapters according to claim 1,wherein at least one modified adapter sequence further comprises arestriction endonuclease cleavage site.
 10. The mixture of 3′ DNAadapters according to claim 1, wherein at least one modified adaptersequence further comprises a PCR primer binding site.
 11. The mixture of3′ DNA adapters of claim 7, wherein the dideoxy nucleotide is ddC. 12.The mixture of 3′ DNA adapters according to claim 1, wherein: at leastone modified adapter sequence AAe comprises AAAe, AACe, AAGe, or AATe;at least one modified adapter sequence ACe comprises ACAe, ACCe, ACGe,or ACTe; at least one modified adapter sequence AGe comprises AGAe,AGCe, AGGe, or AGTe; at least one modified adapter sequence ATe,comprises ATAe, ATCe, ATGe, or ATTe; at least one modified adaptersequence CAe comprises CAAe, CACe, CAGe, or CATe; at least one modifiedadapter sequence CCe comprises CCAe, CCCe, CCGe, or CCTe; at least onemodified adapter sequence CGe comprises CGAe, CGCe, CGGe, or CGTe; atleast one modified adapter sequence CTe comprises CTAe, CTCe, CTGe, orCTTe; at least one modified adapter sequence GAe comprises GAAe, GACe,GAGe, or GATe; at least one modified adapter sequence GCe comprisesGCAe, GCCe, GCGe, or GCTe; at least one modified adapter sequence GGecomprises GGAe, GGCe, GGGe, or GGTe; at least one-modified adaptersequence GTe comprises GTAe, GTCe, GTGe, or GTTe; at least one modifiedadapter sequence TAe comprises TAAe, TACe, TAGe, or TATe; at least onemodified adapter sequence TCe comprises TCAe, TCTe, TCGe, or TCTe; atleast one modified adapter sequence TGe comprises TGAe, TGCe, TGGe, orTGTe; or at least one modified adapter sequence TTe comprises TTAe,TTCe, TTGe, or TTTe.
 13. The mixture of adapters according to claim 12,wherein the random nucleotides of at least one modified adapter sequencefurther comprises an additional A, C, G, or T random nucleotide.
 14. Amixture of 5′ adapters and 3′ adapters comprising: a plurality of 5′modified adapter sequences and a plurality of 3′ modified adaptersequences, wherein the 5′ modified adapter sequences comprise RNA andthe 3′ modified adapter sequences comprise DNA and an activatedriboadenylation (rAPP) at the 5′ end; wherein each modified adaptersequence comprises an adapter sequence and two random nucleotides,wherein the two random nucleotides are located on the 3′ end of the 5′modified adapter sequences and wherein the two random nucleotides arelocated between the activated adenylation and the adapter sequence ofthe 3′ modified adapter sequences; wherein for each 5′ adapter sequenceof unique sequence in the mixture, the mixture comprises sixteenmodified adapter sequences, each comprising the unique adapter sequenceand one of the sixteen possible combinations of two random nucleotides;and wherein for each 3′ adapter sequence of unique sequence in themixture, the mixture comprises sixteen modified adapter sequences, eachcomprising the unique adapter sequence and one of the sixteen possiblecombinations of two random nucleotides.
 15. A mixture of 5′ adapters and3′ adapters comprising: a plurality of 5′ modified adapter sequences anda plurality of 3′ modified adapter sequences, wherein the 5′ modifiedadapter sequences comprise RNA and the 3′ modified adapter sequencescomprise DNA and an activated riboadenylation (rAPP) at the 5′ end;wherein each modified adapter sequence comprises an adapter sequence andthree random nucleotides, wherein the three random nucleotides arelocated on the 3′ end of the 5′ modified adapter sequences and whereinthe three random nucleotides are located between the activatedadenylation and the adapter sequence of the 3′ modified adaptersequences; wherein for each 5′ adapter sequence of unique sequence inthe mixture, the mixture comprises sixty four modified adaptersequences, each comprising the unique adapter sequence and one of thesixty four possible combinations of three random nucleotides; andwherein for each 3′ adapter sequence of unique sequence in the mixture,the mixture comprises sixty four modified adapter sequences, eachcomprising the unique adapter sequence and one of the sixty fourpossible combinations of three random nucleotides.
 16. A mixture of 5′adapters and 3′ adapters comprising: a plurality of 5′ modified adaptersequences and a plurality of 3′ modified adapter sequences, wherein the5′ modified adapter sequences comprise RNA and the 3′ modified adaptersequences comprise DNA and an activated riboadenylation (rAPP) at the 5′end; wherein each modified adapter sequence comprises an adaptersequence and four random nucleotides, wherein the four randomnucleotides are located on the 3′ end of the 5′ modified adaptersequences and wherein the four random nucleotides are located betweenthe activated adenylation and the adapter sequence of the 3′ modifiedadapter sequences; wherein for each 5′ adapter sequence of uniquesequence in the mixture, the mixture comprises two hundred and fifty sixmodified adapter sequences, each comprising the unique adapter sequenceand one of the two hundred and fifty six possible combinations of fourrandom nucleotides; and wherein for each 3′ adapter sequence of uniquesequence in the mixture, the mixture comprises two hundred and fifty sixmodified adapter sequences, each comprising the unique adapter sequenceand one of the two hundred and fifty six possible combinations of fourrandom nucleotides.
 17. The mixture of 3′ DNA adapters according toclaim 1, wherein the at least two random nucleotides comprisedeoxyguanylate, deoxyadenylate, thymidylate, deoxycytidylate, guanylate,adenylate, uridylate, cytidylate, deoxyinosine monophosphate, inosinemonophosphate, deoxy-5-bromouridylate or 5-bromouridylate.